Oligonucleotide-mediated, site-directed mutagenesis was developed in the 1970's in bacteriophage .phi.X174 (see, e.g., Weisbeek and Van de Pol); Hutchinson and Edgell (1971)) and in E. coli (see, e.g., Hutchinson et al. (1978); Razin et al. (1978)) as a means of introducing specific, predetermined changes or "mutations" at specific, predetermined sites in DNA molecules replicated in vivo. Subsequently, in vitro techniques for oligonucleotide-mediated mutagenesis have become routine in the fields of molecular genetics and biotechnology to introduce mutations into relatively long nucleic acid molecules (see, e.g., Sambrook et al. (1989)).
In brief, these techniques depend upon the facts that (1) under appropriate conditions, imperfectly complementary nucleic acids of sufficient length are capable of hybridizing to form heteroduplexes with mismatched or non-complementary base pairings, and (2) template-dependent polymerase-mediated nucleic acid synthesis proceeds 5'.fwdarw.3' from "primer" sequences duplexed to a template sequence. Therefore, to introduce a change or mutation into a relatively long sequence, a relatively short oligonucleotide may be prepared which includes the desired mutation and which will (1) hybridize to a complementary portion of the template sequence, and (2) serve as a mutagenic primer to support template-dependent polymerase-mediated synthesis of the relatively longer nucleic acid bearing the desired mutation. In practice it has been shown that single or multiple mutations may be introduced to nucleic acids of hundreds or thousands of nucleotides using template nucleic acids and one or more mutagenic oligonucleotide primers in vivo or in vitro.
In molecular biology and biotechnology, oligonucleotide-mediated mutagenesis can be used, inter alia, to introduce mutations in protein-encoding nucleic acids to introduce, remove or alter (1) one or a few amino acids in the encoded polypeptide products, (2) genetic regulatory sites (e.g., start or stop codons, promoter or enhancer sequences, polyadenylation sites, intron-exon splicing sites), and (3) restriction enzyme cleavage sites; as well as to create (4) large pools or libraries of combinatorially differing nucleic acid sequences which can be used to create corresponding pools or libraries of combinatorially differing polypeptides.
More recently, nucleic acids have been investigated for their potential utility in the computational sciences because, inter alia, their nucleotide sequences provide a molecular-scale medium for information storage, and because those sequences may be manipulated by the various means known and employed in the biological sciences (e.g., restriction, ligation, polymerase-dependent amplification and oligonucleotide-mediated mutagenesis). Notably, Adelman (1994, 1995) and others have used nucleic acid molecules to encode information and have used the reactions of large populations of nucleic acids in solution as massively parallel processors to find the solution to "Hamiltorian-path" problems.
Prior to the present invention, however, no method or products have been described for the introduction of mutations in a programmed sequential manner, in which one mutation causes base changes that enable later mutations. As described herein, such methods and products have utilities in both the biological and computational sciences.